Expression of dengue virus and Zika virus NS2B-NS3pro constructs alter cellular fatty acids, but co-expression with a Zika virus virus-like particle is detrimental to virus-like particle expression

Objective Studies have shown that Flavivirus infection remodels the host cell to favour viral replication. In particular, the host cell lipid profile is altered, and it has been proposed that this process alters membrane fluidity to allow wrapping of the outer structural proteins around the viral nucleocapsid. We investigated whether expression of the Zika virus (ZIKV) and dengue virus (DENV) protease induced alterations in the cellular lipid profile, and subsequently whether co-expression of these proteases with VLP constructs was able to improve VLP yield. Results Our results showed that both ZIKV and DENV proteases induced alterations in the lipid profile, but that both active and inactive proteases induced many of the same changes. Neither co-transfection of protease and VLP constructs nor bicistronic vectors allowing expression of both protease and VLP separated by a cell cleavable linker improved VLP yield, and indeed many of the constructs showed significantly reduced VLP production. Further work in developing improved VLP expression platforms is required. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-023-06572-z.


Additional method
Plasmid constructs DENV NS2B-NS3pro and NS2B-NS3(S135A) were constructed using cDNA of DENV 2 (strain 16681) as a template.The 48 amino acid central portion of the NS2B precursor protein (residues 49-96) and 185 amino acids of NS3 protease precursor protein (residues 1-185) were linked in frame with a Gly4SerGly4 linker (GGGGSGGGG) and cloned in frame with octahistidine (8xHis) tag at 3′ end of NS3 protease.The fragment of the NS2B cofactor linked with a Gly4SerGly4 was

Lipid extraction
Lipids were extracted from cell pellets by a modified Bligh and Dyer protocol [Bligh and Dyer, 1959] using a ratio of Chloroform: Methanol: H2O of 2: 2: 1.Briefly, 160 µl of chloroform (HPLC grade) was added to cells and then samples were incubated on ice for 1 h.After incubation, 320 µl of methanol (UHPLC grade) were added and tubes were agitated at 600 rpm at 15ºC for 30 min.
After that, 150 µl of water (HPLC grade) was added and samples were further agitated at 600 rpm at 15ºC for 30 min.Finally, a further 160 µl of chloroform was added to the samples which were then agitated for 10 min before centrifugation at 13,500xg at 15ºC for 10 min.After centrifugation, the lower organic phase that contained of lipids was removed and transferred into a 3 ml furnace glass vial.The remaining aqueous phase was re-extracted once more with chloroform before centrifugation at 13,500xg at 15ºC for 10 min.The lower organic phase was removed and pooled with the previous extraction.All lipid samples were dried under a gentle stream of nitrogen until the sample had evaporated.

Fatty acid profile using GC/Q-TOF
A total of 50 µl of myristic acid D27 (500 ppm in hexane) as an internal standard (IS) was added to the dried sample (from 100 µl of the lower organic phase at last step of the lipid extraction).
The vial containing sample and IS was dried under vacuum at 60ºC for 30 min to remove the residual solvent.Subsequently 1% sulfuric acid in methanol (500 ul) was added to the vial, and the esterification reaction was initiated by incubation at 50 ºC for 2h.After that the vial was cooled to room temperature followed by the addition of 500 l of 15% NaCl and 500 l of hexane.The solution was mixed for 5 min using a multichannel mixer at 2400 rpm.After mixing, the samples were centrifuged at 3,000 x g for 5 min.The 200 l-hexane supernatant layers were transferred into 1.5 ml V-shaped GC vials for GC analysis.
The samples were analyzed using a gas chromatography-quadrupole time of flight mass spectrometer (GC/Q-TOF, GC 7890B/MSD 7250, Agilent Technologies, USA) coupled to a PAL auto sampler system (CTC Analytics AG, Switzerland).An aliquot of the derivatized samples (1 µl) was injected into the GC/Q-TOF using a pulsed split mode with an injector temperature of 250°C, 45 psi until 0.5 min, a split ratio of 500 to 1, and a CP-Sil-88 column (100 m, 0.25 mm i.d., 0.20 m film, Agilent Technologies, USA).Helium was used as the carrier gas with a constant flow rate of 1.2 ml/min.The GC oven was programmed as follows: The initial oven temperature was controlled at 100 °C, help for 0.5 min.Then, the temperature was ramped from 100 °C to held for 5 min, finally ramped from 185 °C to 230 °C at the rate of 10 °C/min, and held for 20 min.Total run time was 39.5 min.The transfer line, ion source (EI), and quadrupole were set as 250 °C, 240 °C, and 150 °C, respectively.The mass spectrometer was operated in full scan mode, ranging from m/z 20-1200 with a data acquisition rate of 5 Hz.MS data was acquired using MassHunter software (version 10.0, Agilent Technologies, USA) utilizing three independent biological replicates to calculate the mean and the standard error.
The calibration curves were generated by mixing an equal volume of the mixture of fatty acid methyl esters (20 to 1,000 ppm) myristic acid-D27 methyl ester in hexane.Myristic acid-D27 methyl ester was prepared using the same method as described above; however, the concentration was increased twice in order to obtain the same concentration of the internal standard in the final Supplemental Table S1.Primers used to develop bicistronic vectors Fragment Primer F Sequence Supplemental Table S3
mixture.Quantitative analyses were performed on Agilent MassHunter software (version 10.0 Agilent Technologies, USA), and exported into Microsoft Excel for further data processing.Supplemental Figure S1.Light and Fluorescent microscopy of transfected cells.HEK293T/17 cells were co-transfected plasmids containing a ZIKV VLP and a plasmid expressing EGFP, or transfected with a bicistronic vector expressing EGFP and a ZIKV VLP separated by a cleavable linker.On day 3 post-transfection cells were examined under a fluorescent microscope.Supplemental Figure S2.DENV FAME Volcano plot.Left: Data from DNS2B-NS3pro (a functional protease) transfected cells compared with mock transfected cells and Right: Data from DNS2B-NS3 (S135A) (a non-functional protease) transfected cells compared with mock transfected cells.Color by p-value fold change cut-offs: gray color represents failure to pass both cut-offs (not significant), blue color represents passing both cut-offs and is down regulated while red color represents the result passed both cut-offs and is up regulated.Supplemental Figure S3.ZIKV FAME Volcano plot.Left: Data from ZNS2B-NS3pro (a functional protease) transfected cells compared with mock transfected cells and Right: Data from ZNS2B-NS3 (S135A) (a non-functional protease) transfected cells compared with mock transfected cells.Color by p-value fold change cut-offs: gray color represents failure to pass both cut-offs (not significant), blue color represents passing both cut-offs and is down regulated while red color represents the result passed both cut-offs and is up regulated.Supplemental Figure S4.Schematic of bicistronic expression plasmids.Seven bicistronic expression vectors were constructed.Each construct contains the ZIKV VLP downstream of either EGFP or ZIKV NS2B-NS3pro, ZIKV NS2B-NS3pro (S135A), ZIKV NS3pro, DENV NS2B-NS3pro, DENV NS2B-NS3pro (S135A) or DENV NS3pro separated by a cell cleavable linker (P2A).The constructs are translated as a single polypeptide (B) which generates a (C) separate VLP after cell cleavage.Supplemental Figure S5.ZIKV VLP analysis HEK293T/17 cells were transfected with either (A) mock or a (B) ZIKV VLP (ZVLP) or a bicistronic plasmid vectors containing a ZIKV VLP downstream of either expressing (C) ZIKV NS3pro (ZNS3), or (D) DENV NS3pro (DNS3) that was cleaved to an ZNS3 or DNS3 and ZVLP.At 3 days post transfection the supernatant was collected, concentrated and purified by discontinuous sucrose gradient.ZIKV VLP particles were then observed by transmission electron microscopy (TEM, HT7700 Hitachi) with magnification 30,000X.Supplemental Figure S6.Quantitation of ZIKV NS3 expression from co-transfection and bicistronic constructs.

Table S4 . Differentially regulated FA compounds by active DENV protease.
Fatty acid compounds differentially regulated by DNS2B-NS3pro as compared to mock.The result show down regulated compounds which passed both cut-offs (there were no upregulated compounds).Compounds in bold are common between active and inactive DENV NS2B-NS3pro constructs.

Table S5 . FA compounds differentially regulated by inactive DENV protease. Fatty acid
compounds differentially regulated by DNS2B-NS3pro(135A) as compared to mock.The result show compounds down regulated and up regulated which passed both cut-offs.Compounds in bold are common between active and inactive DENV NS2B-NS3pro constructs.

Table S7 . FA compounds differentially regulated by inactive ZIKV protease.
Fatty acid compounds differentially regulated byZNS2B-NS3 (135A) as compared to mock.The result show compounds down regulated and up regulated which passed both cut-offs.Compounds in bold are common between active and inactive ZIKV NS2B-NS3pro constructs.